We have demonstrated that clinical isolates of Staphylococcus aureus are more virulent than the prototype laboratory strain (RN6390) in our animal models of musculoskeletal infection. We have confirmed that the genomes of these isolates includes genes that are not present in RN6390, and we hypothesize that there is a subset of genes that defines musculoskeletal isolates from their less virulent counterparts. We will test this hypothesis in Aim 1. Additionally, we have established that regulatory circuits in clinical isolates are distinct by comparison to RN6390 both in terms of the wild-type strains themselves and their respective agr and sarA mutants. Our studies indicate that this is due to a regulatory imbalance in RN6390 that results in a phenotype dominated by agr. In contrast, the phenotype of clinical isolates is dominated by sarA. An important aspect of this is that clinical isolates have an enhanced capacity to form a biofilm. Consistent with this hypothesis is our demonstration that mutation of sarA results in a reduced capacity to form a biofilm. Importantly, that is true in all S. aureus strains other than RN6390. Based on this, we hypothesize that specific components of the sarA regulon are required for biofilm formation and/or adaptation to the sessile lifestyle. In Aim 2, we will test this hypothesis by correlating the transcriptional profile of the relevant sarA mutants with the profile observed in bacteria harvested from biofilms. This will be done using comprehensive microarrays representing the genomes of all seven of the sequenced strains of S. aureus as well as the virulent clinical isolates themselves. Finally, we have also confirmed that mutation of sarA also limits the ability of our clinical isolates to cause disease, and we hypothesize that the inability to form a biofilm may be responsible, at least in part, for this attenuation. In Aim 3, we will test this hypothesis by correlating the ability to form a biofilm with virulence in our infection models. To accomplish these goals, we will 1) determine whether the genome of S. aureus strains that cause musculoskeletal infection includes a subset of genes that contributes to their virulence and is absent in less virulent laboratory strains, 2) correlate transcriptional changes associated with mutation of sarA with the adaptive response required for persistence within a biofilm and 3) define the impact of genes identified in Aims 1 and 2 on biofilm formation and virulence by generating appropriate mutations in clinical isolates of S. aureus and evaluating the impact using in vivo models of biofilm formation and musculoskeletal disease. We believe these experiments will ultimately lead to the identification of novel therapeutic targets for the treatment and prevention of staphylococcal musculoskeletal infection. [unreadable] [unreadable]